The present invention relates to a method of identifying amino acids, xcex1-hydroxyketones and hexose phosphates in the blood spots of newborns using elecrtospray tandem mass spectrometry. The present invention further relates to a novel method using a diimine metal ligand.
Tandem mass spectrometry (MS/MS) has been used for several years to identify and measure carotene esters in blood and urine of children suspected of having inborn errors of metabolism. More recently, MS/MS has been used in pilot programs to screen newborns for conditions and disorders of amino and organic acids metabolism as well. A general overview of mass spectrometry, and in particular the tandem mass spectrometry and its use in newborn screening, can be found in xe2x80x9cTandem Mass Spectrometry in Newborn Screeningxe2x80x9d (ACGM/ASHG statement; Genetics and Medicine, July/August 2000, Volume 2, No. 4, page 267-269).
The current methodologies for tandem mass spectrometry screening use either butyl esters of the amino acids and acyl carnitine (Rashed M. S. et al. Sernin 1999: 25(2): 183-93) or direct underivatized analysis. The former causes degradation of some amino acid species and is very time consuming, and the latter suffers from insensitivity for some important amino acids.
Electrospray ionization of transition metal diimine ligand: xcex1 amino acid complexes has been demonstrated to provide sensitive determination of amino acid constituents of proteins in standard solutions (Gatlin, C. L. et al. J. Mass Spectrom 1995; 30: 1605-1616 and Gatlin, C. L. et al. J. Mass Spectrom 1995; 30: 1617-1627). However, there is a problem using this method in identifying homocystine or homocysteine to the same extent as the other amino acids. Thus, there is a need to improve the sensitivity of this method. In addition there is a need to extend the use of tandem mass spectrometry to include the identification of other compounds besides amino acids in the analysis. For example, congenital adrenal hyperplasia (CAH), a disorder caused by deficiency of the 21-hydrolase enzyme, is the most common inborn error of the adrenal steroid pathways. Early diagnosis and monitoring of CAH can be life saving. Monitoring and screening for CAH patients by measuring levels of 17 xcex1-hydroxyprogesterone (17 OHP) or other steroids, has become a routine part of many programs. Numerous methods have been described to determine these steroid hormones, such as flourometry, radio immunoassay and high-performance liquid chromotography. A method for detecting 17 OHP using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry was described by Lai et al. (Rapid Communication in Mass Spectrometry, 2001,15: 2145-2151) and Shindo et al. (Biomedical Chromatography, 1990, 4:171-174). Most of these methods, however, are affected by a degree of interference or cross-reactivity with other steroids.
In a further application, galactosemia is an inherited disorder wherein the metabolism of galacatose caused by a deficiency of the enzyme galacatose-1 phosphate uridyl transferase. Galactosemia leads to accumulation of galacatose and galacatose-1 phosphate in blood and tissue and if left untreated, can result in neonatal death, or severe mental retardation, pyrosis of the liver and cardiacs. There are several available methods for neonatal screening for galactosemia. The simplest involves examining the urine for reducing substances. Other methods include activity assays for the enzyme galacatose-1 phosphate uridyl transferase and microbiological assays screening for galactose and galactose-1 phosphate. A method using tandem mass spectrometry is described by Jensen et al. (Clinical Chemistry, 2001, 74: 1364-1372.)
With the tandem mass spectrometry methods used to date, there is no single method that can be used to identify amino acids, xcex1-hydroxyketones and hexose phosphates. This need is addressed in the present invention.
The present invention relates to a method of identifying amino acids, xcex1-hydroxyketones and hexose phosphates in the blood spots of newborns using elecrtospray tandem mass spectrometry. The present invention further relates to a novel method using a diimine metal ligand.
Thus, according to the present invention, there is provided a method of new born screening for selected analytes using tandem mass spectrometry comprising the steps of: mixing a sample of blood or blood spot eluate with a metal diimine compound to form a metal diimine complex; subjecting said complex top high performance liquid chromatography to produce an eluate; subjecting the eluate from the HPLC step to an electrospray ion source of a mass spectrometer to yield MS/MS product ions; and detecting the MS/MS product ions.